肺纤维化Caveolin1 泛素化降解机制和氟非尼酮治疗靶点的研究

Pulmonary fibrosis is a disease which does great harm to human health, with complicated pathological mechanism and poor response to present treatments. Caveolin 1(CAV1) is suggested to play a critical role in the pathogenesis of pulmonary fibrosis according to researches, by its own downregulation and regulation of various signal pathways. But the reason of its downregualtion in fibrosis is still unknown, and upregulation of CAV1 is thought to be an ideal therapeutic strategy. Fluorofenidone (AKF-PD) is novel anti-fibrotic medicine developed by our research group, which has been applied for the clinicalⅠtrials. Previous studies showed that it could increase the expression of CAV1 and had a therapeutic effect to pulmonary fibrosis. Further study found that AKF-PD could also inhibit the degradation of CAV1 while do not affect its synthesis. Based on these findings, we assume that abnormality of CAV1 ubiquitination may be an important mechanism of pulmonary fibrosis, and AKF-PD can protect from pulmonary fibrosis by inhibiting it. We intend to use CAV1-/- and ubiquitin ligase E3 Cbl-b-/- mice and cells as subjects, to investigate whether accelerated ubiquitination is the main reason of downregulation of CAV1 in pulmonary fibrosis, and whether ubiquitin ligase E3 Cbl-b plays an important role in this process, and be an effective target of AKF-PD in the treatment of pulmonary fibrosis.

肺纤维化严重危害人类健康，其发病机制复杂，治疗困难。研究认为，小窝蛋白1(CAV1)表达下降和其调控的多条信号通路异常，在肺纤维化发病中起重要作用；但CAV1表达下调的机制有待阐明，而上调CAV1是治疗肺纤维化的理想策略。氟菲尼酮(AKF-PD)是本课题组发明的新型抗纤维化药物，已经申请进入国家1类新药Ⅰ期临床试验。前期研究发现，AKF-PD能够提高CAV1、治疗肺纤维化；进一步发现AKF-PD抑制CAV1的降解，但不促进CAV1合成。据此我们设想：CAV1泛素化降解异常是肺纤维化的重要机制，AKF-PD通过抑制CAV1泛素化降解而起抗肺纤维化作用。本项目将利用CAV1-/-小鼠、泛素连接酶Cbl-b-/-小鼠和细胞模型开展研究，以探明CAV1泛素化加速是肺纤维化CAV1表达减少的主要原因，泛素连接酶Cbl-b在CAV1降解中起重要作用，CAV1泛素化是AKF-PD治疗肺纤维化的作用靶点。

肺纤维化严重危害人类健康，其发病机制复杂，治疗困难。研究认为，小窝蛋白1(CAV1)表达下降和其调控的多条信号通路异常，在肺纤维化发病中起核心作用；但 CAV1表达下调的机制有待阐明，而上调 CAV1 是治疗肺纤维化的理想策略。氟菲尼酮(AKF-PD)是本课题组发明的新型抗纤维化药物，已经获批进入国家1.1类新药Ⅰ期临床试验。课题组前期研究发现，氟非尼酮具有抗肺纤维化的作用，能提高博来霉素肺纤维化小鼠肺组织CAV1 表达和通过上调正常原代人肺成纤维细胞（NHLFs）中CAV1的表达抑制NHLFs活化，但其具体机制尚不清楚。本研究在细胞和动物水平，综合运用GST-pulldown , 沉默/过表达、免疫沉淀、基因敲除等研究手段，阐明了：（1）氟非尼酮可通过提高Cav1蛋白表达、抑制PTEN/PI3K/Akt通路减轻肺纤维化和成纤维细胞活化。（2）氟非尼酮能够抑制TGF-β1介导的Cav1蛋白泛素化降解，从而提高Cav1蛋白表达。（3）Parkin在NHLFs细胞中可以作为Cav1的E3连接酶促进Cav1泛素化降解；氟非尼酮不直接改变Parkin表达，可能是通过抑制Parkin和Cav1的相互作用抑制Cav1泛素化降解。在本课题中，我们进一步明确了氟非尼酮通过抑制Cav1泛素化降解、提高Cav1蛋白表达，从而抑制PTEN/PI3K/Akt通路减轻成纤维细胞活化，加深了我们对氟非尼酮抗肺纤维化机制的理解，也为抗间质纤维化药物的开发提供新的方向。